Conjugate of an antibody against CCR5 and an antifusogenic peptide

ABSTRACT

The current invention is related to a conjugate comprising one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) characterized in that one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody and to the pharmaceutical use of said conjugate.

PRIORITY

This application claims priority under 35 USC §119 from EuropeanApplication EP06017156.8 filed Aug. 17, 2006, and EP06020647.1 filedSep. 29, 2006, both of which are incorporated herein by reference infull.

FIELD OF THE INVENTION

The present invention relates to a conjugate of an antibody against CCR5and an anti-fusogenic peptide wherein one to eight antifusogenicpeptides are each conjugated to one terminus of the heavy and/or lightchains of an anti-CCR5 antibody. The antifusogenic peptides can bedifferent, similar or identical on the amino acid level.

BACKGROUND OF THE INVENTION

The infection of cells by the human immunodeficiency virus (HIV) iseffected by a process in which the membrane of the cells to be infectedand the viral membrane are fused. A general scheme for this process isproposed: The viral envelope glycoprotein complex (gp120/gp41) interactswith a cell surface receptor located on the membrane of the cell to beinfected. The binding of gp120 to, e.g., the CD4 receptor in combinationwith a co-receptor such as CCR-5 or CXCR-4 causes a change in theconformation of the gp120/gp41 complex. In consequence of thisconformational change the gp41 protein is able to insert into themembrane of the target cell. This insertion is the beginning of themembrane fusion process. It is known that the amino acid sequence of thegp41 protein varies in different HIV strains because of naturallyoccurring polymorphisms. But the same domain architecture can berecognized, more precisely, a fusion signal, two heptad repeat domains(HR1, HR2) and a transmembrane domain (in N- to C-terminal direction).It is suggested that the fusion (or fusogenic) domain is participatingin the insertion into and the disintegration of the cell membrane. TheHR regions are built up of multiple stretches comprising seven aminoacids (“heptad”) (see e.g. W. Shu, et al., Biochem (1999) 38:5378-85).Beside the heptads one or more leucine zipper-like motifs are present.This composition accounts for the formation of a coiled coil structureof gp41 proteins and just as well of peptides derived from thesedomains. Coiled coils are in general oligomers consisting of two or moreinteracting helices. Peptides with amino acid sequences deduced from theHR1 or the HR2 domain of gp41 are effective in vitro and in vivoinhibitors of HIV uptake into cells (for example peptides see e.g. U.S.Pat. No. 5,464,933, U.S. Pat. No. 5,656,480, U.S. Pat. No. 6,258,782,U.S. Pat. No. 6,348,568, or U.S. Pat. No. 6,656,906). For example, T20(also known as DP178, Fuzeon®, a HR2 peptide), T651 (U.S. Pat. No.6,479,055), and T2635 (WO 2004/029074) are very potent inhibitors of HIVinfection. It has been attempted to enhance the efficacy of HR2 derivedpeptides with, for example, amino acid substitutions or chemicalcrosslinking (S. K. Sia, et al., Proc. Natl. Acad. Sci. USA (2002)99:14664-69; A. Otaka, et al., Angew. Chem. Int. Ed. (2002) 41:2937-40).

The conjugation of peptides to certain molecules can change theirpharmacokinetic properties, e.g. the serum half-life of such peptideconjugates can be increased. Conjugations are reported, for example, forpolyethylene glycol (PEG) and Interleukin-6 (EP 0 442 724), for PEG andErythropoietin (WO 01/02017), for chimeric molecules comprisingEndostatin and immunoglobulins (US 2005-008649), for secreted antibodybased fusion proteins (US 2002-147311), for fusion polypeptidescomprising albumin (US 2005-0100991; human serum albumin U.S. Pat. No.5,876,969), for PEGylated polypeptides (US 2005-0114037), and forinterferon fusions. Also described in the state of the art areimmunotoxins comprising Gelonin and an antibody (WO 94/26910), modifiedtransferrin-antibody fusion proteins (US 2003-0226155),antibody-cytokine fusion proteins (US 2003-0049227), and fusion proteinsconsisting of a peptide with immuno-stimulatory, membrane transport, orhomophilic activity and an antibody (US 2003-0103984). In WO 2004/085505long acting biologically active conjugates consisting of biologicallyactive compounds chemically linked to macromolecules, are reported.

The co-receptor CCR5 is used by most HIV-1 primary isolates and iscritical for the establishment and maintenance of infection. Inaddition, CCR5 function is dispensable for human health. A mutant CCR5allele, “CCR5Δ32”, encodes a truncated, non-functional protein (M.Samson, et al., Nature (1996) 382:722-25; M. Dean, et al., Science(1996) 273:1856-62). Individuals homozygous for the mutation lack CCR5expression and are strongly protected from HIV-1 infection. Theydemonstrate no overt phenotype consequence and are highly resistant toM-tropic HIV infection, whereas heterozygote individuals present delayeddisease progression (M. K. Schwarz and T. N. Wells, Nat. Rev. DrugDiscov. (2002) 1:347-58). The lack of CCR5 is without apparent adverseconsequences, probably because CCR5 is part of a highly redundantchemokine network as receptor for the α-chemokines MIP-1α, MIP-1β, andRANTES, which share many overlapping functions, and most of which havealternative receptors (D. Rossi and A. Zlotnik, Ann. Rev. Immunol.(2000) 18:217-42). The identification of CCR5 as an HIV-1 co-receptorwas based on the ability of its ligands, MIP-1α, MIP-1β, and RANTES, toblock infection by R5 but not R5X4 or X4 isolates (F. Cocchi, et al.,Science (1995) 270:1811-15). CCR5 is also a receptor of the “cluster”chemokines, which are produced primarily during inflammatory responsesand control the recruitment of neutrophils (CXC chemokines), macrophagesand a subset of T cells (T helper Th1 and Th2 cells). Th1 responses aretypically those involving cell-mediated immunity effective againstviruses and tumors, proinflammatory responses responsible for killingintracellular parasites, and perpetuating autoimmune responses, forexample, whereas Th2 responses are believed to be pivotal in allergies.Therefore, inhibitors of these chemokine receptors may be useful asimmunomodulators. For Th1 responses, overactive responses are dampened,for example, in autoimmunity including rheumatoid arthritis, or, for Th2responses, asthma attacks or allergic responses including atopicdermatitis are lessened (see e.g. D. Schols, Curr. Top. Med. Chem.(2004) 4:883-93; A. Mueller and P. G. Strange, Int. J. Biochem. CellBiol. 36:35-38; W. M. Kazmierski et al., Curr. Drug Targets Infect.Disord. (2002) 2:265-78; T. Lehner, Trends Immunol. (2002) 23:347-51).

Antibodies against human CCR5 are e.g. PRO140 (W. C. Olson et al., J.Virol. (1999) 73:4145-55), and/or 2D7 (M. Samson et al., J. Biol. Chem.(1997) 272:24934-41). Additional antibodies are mentioned in US2004-0043033, U.S. Pat. No. 6,610,834, US 2003-0228306, US 2003-0195348,US 2003-0166870, US 2003-0166024, US 2003-0165988, US 2003-0152913, US2003-0100058, US 2003-0099645, US 2003-0049251, US 2003-0044411, US2003-0003440, U.S. Pat. No. 6,528,625, US 2002-0147147, US 2002-0146415,US 2002-0106374, US 2002-0061834, US 2002-0048786, US 2001/0000241, EP 1322 332, EP 1 263 791, EP 1 207 202, EP 1 161 456, EP 1 144 006, WO2003/072766, WO 2003/066830, WO 2003/033666, WO 2002/083172, WO02/22077, WO 01/58916, WO 01/58915, WO 01/43779, WO 01/42308, and EP05007138.0.

Polyethylene glycol conjugates of antibodies against CCR5 are known fromUS 2003-0228306. US 2003-0215421 refers to chemokine-toxin conjugates.WO 01/43779 refers to conjugates of anti-CD4 antibodies and anti-CCR5antibodies and to conjugates of anti-CD4 antibodies and an HIV-1 fusioninhibiting peptide. Conjugates of CCR5 antibodies and toxins arementioned in EP 1 346 731.

SUMMARY OF THE INVENTION

The invention comprises a conjugate comprising one or more antifusogenicpeptides and an anti-CCR5 antibody (mAb CCR5) characterized in that oneto eight antifusogenic peptides are each conjugated to one terminus ofthe heavy and/or light chains of said anti-CCR5 antibody (a number ofeight antifusogenic peptides per mAb CCR5 is only possible if the mAbCCR5 comprises eight termini, i.e. is composed e.g. of two heavy chainsand two light chains; if the mAb CCR5 comprises a smaller number of C-and N-termini, e.g. as a scFv, the corresponding number of antifusogenicpeptides possible at maximum in the conjugate is also reduced, i.e. itis reduced to less than eight).

Preferably the carboxy-terminal amino acid of an anti-CCR5 antibodychain is conjugated to the amino-terminal amino acid of theantifusogenic peptide or the carboxy-terminal amino acid of theantifusogenic peptide is conjugated to the amino-terminal amino acid ofthe antibody chain, preferably by a peptide bond with or without anintermediate linker.

Preferably the conjugate is characterized by the general formula

mAb CCR5-[linker]_(m)-[antifusogenic peptide]_(n)

wherein m is independently for each antifusogenic peptide either 0 (i.e.a peptide bond between mAb CCR5 and antifusogenic peptide) or 1 (i.e. alinker between mAb CCR5 and antifusogenic peptide) and n is an integerof from 1 to 8.

A preferred conjugate of a heavy and/or light chain of mAb CCR5 and anantifusogenic peptide (“chain conjugate”) is selected from the groupconsisting of:

-   -   (1) [antifusogenic peptide]-[linker]_(m)-[heavy chain]    -   (2) [heavy chain]-[linker]_(m)-[antifusogenic peptide]    -   (3) [antifusogenic peptide]-[linker]_(m)-[heavy        chain]-[antifusogenic peptide]    -   (4) [antifusogenic peptide]-[linker]_(m)-[light chain]    -   (5) [light chain]-[linker]_(m)-[antifusogenic peptide]    -   (6) [antifusogenic peptide]-[linker]_(m)-[light        chain]-[antifusogenic peptide]    -   (7) [antifusogenic peptide]-[linker]_(m)-[heavy        chain]-[linker]_(m)-[antifusogenic peptide]    -   (8) [antifusogenic peptide]-[linker]_(m)-[light        chain]-[linker]_(m)-[antifusogenic peptide]        wherein the linker can be the same or different in (within and        between) said chain conjugates, wherein m is an integer of 1 or        0, and m can be independently the same or different in (within        and between) said chain conjugates.

(“Left side” of the peptide or mAb CCR5 chain means N-terminus, “rightside” means C-terminus. In (1) therefore the C-terminus of theantifusogenic peptide is linked by a peptide bond or a linker to theN-terminus of the heavy chain of mAb CCR5).

Preferably the chain conjugates are assembled to conjugates according tothe invention comprising a mAb CCR5 (e.g. consisting of two light chainsand two heavy chains including the constant Fc domains, a scFv fragment,or a Fab fragment).

Especially preferred chain conjugates are (2), (3), (4), and (7).Especially preferred conjugates according to the invention comprise2×[mAb CCR5 light chain] and 2×(2), 2×[mAb CCR5 light chain] and 2×(3),or 2×[mAb CCR5 heavy chain] and 2×(4), or 2×[mAb CCR5 light chain] and2×(7). The heavy and/or light chain comprises preferably a constantregion (Fc).

Preferably the conjugate is characterized in comprising a variable heavychain domain consisting of an immunoglobulin framework and a CDR3 regionselected from the group consisting of the heavy chain CDR3 sequences SEQID NO: 16, 17.

Preferably the conjugate is characterized in comprising a variable heavychain domain consisting of an immunoglobulin framework and a CDR3 regionselected from the group consisting of CDR3 sequences SEQ ID NO: 16, 17,a CDR2 region selected from the group consisting of CDR2 sequences SEQID NO: 13, 14, 15, and a CDR1 region selected from the group consistingof CDR1 sequences SEQ ID NO: 9, 10, 11, 12.

Preferably the conjugate is characterized in comprising a heavy chainvariable domain selected from the group of heavy chain variable domainscomprising SEQ ID NO: 1, 3, 5, and 7.

Preferably the conjugate is characterized in comprising a variable lightchain domain consisting of an immunoglobulin framework and a CDR1 regionselected from SEQ ID NO:18, 19, 20, a CDR2 region selected from SEQ IDNO:21, 22, 23, and a CDR3 region selected from SEQ ID NO:24, 25.

Preferably the conjugate is characterized in comprising as heavy chainCDRs the CDRs of SEQ ID NO:1 and as light chain CDRs the CDRs of SEQ IDNO:2, as heavy chain CDRs the CDRs of SEQ ID NO:3 and as light chainCDRs the CDRs of SEQ ID NO:4, as heavy chain CDRs the CDRs of SEQ IDNO:5 and as light chain CDRs the CDRs of SEQ ID NO:6, or as heavy chainCDRs the CDRs of SEQ ID NO:7 and as light chain CDRs the CDRs of SEQ IDNO:8.

Preferably the conjugate is characterized in comprising a variable heavyand light chain domain independently selected from the group consistingof

-   a) the heavy chain (V_(H)) variable domain defined by amino acid    sequence SEQ ID NO: 1 and the light chain (V_(L)) variable domain    defined by amino acid sequence SEQ ID NO:2;-   b) the heavy chain variable domain defined by amino acid sequence    SEQ ID NO:3 and the light chain variable domain defined by amino    acid sequence SEQ ID NO:4;-   c) the heavy chain variable domain defined by amino acid sequence    SEQ ID NO:5 and the light chain variable domain defined by amino    acid sequence SEQ ID NO:6;-   d) the heavy chain variable domain defined by amino acid sequence    SEQ ID NO:7 and the light chain variable domain defined by amino    acid sequence SEQ ID NO:8.

Preferably the conjugate is characterized in comprising the heavy chain(V_(H)) variable domain defined by amino acid sequence SEQ ID NO:1 andthe light chain (V_(L)) variable domain defined by amino acid sequenceSEQ ID NO:2; or the heavy chain variable domain defined by amino acidsequence SEQ ID NO:3 and the light chain variable domain defined byamino acid sequence SEQ ID NO:4; or the heavy chain variable domaindefined by amino acid sequence SEQ ID NO:5 and the light chain variabledomain defined by amino acid sequence SEQ ID NO:6; or the heavy chainvariable domain defined by amino acid sequence SEQ ID NO:7 and the lightchain variable domain defined by amino acid sequence SEQ ID NO:8; alinker selected from the group consisting of the amino acids glycine (G)and asparagine (N), the tripeptide GST, and SEQ ID NO:36-62; and anantifusogenic peptide selected from the group of peptides defined by SEQID NO:29 to 35.

Preferably the conjugate is characterized in comprising an antifusogenicpeptide selected from the group of peptides comprising C34, T20, T1249,T651, T2635, N36, and DP107.

Preferably the conjugate is characterized in comprising an antifusogenicpeptide at each C-terminus of the heavy chains or at each N-terminus ofthe light chains (two antifusogenic peptides). Preferably the conjugateis characterized in that it comprises an antifusogenic peptide at eachC-terminus of the heavy chains and at each N-terminus of the lightchains (four antifusogenic peptides).

Preferably the conjugate is characterized in comprising two light chainvariable domains of SEQ ID NO:2, two conjugates of type (2) eachcomprising a heavy chain variable domain of SEQ ID NO:1, a linker of SEQID NO:40 and an antifusogenic peptide of SEQ ID NO:33, in comprising twolight chain variable domains of SEQ ID NO:4, two conjugates of type (2)each comprising a heavy chain variable domain of SEQ ID NO:3, a linkerof SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, incomprising two light chain variable domains of SEQ ID NO:6, twoconjugates of type (2) each comprising a heavy chain variable domain ofSEQ ID NO:5, a linker of SEQ ID NO:40 and an antifusogenic peptide ofSEQ ID NO:33, or in comprising two light chain variable domains of SEQID NO:8, two conjugates of type (2) each comprising a heavy chainvariable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and anantifusogenic peptide of SEQ ID NO:33.

Preferably the conjugate is characterized in that said anti-CCR5antibody is of IgG1 subclass. It is also preferred, that said anti-CCR5antibody is of IgG4 subclass, or of IgG1 or IgG2 subclass, with amutation in amino acid position S228, L234, L235, and/or D265, and/orcontains the PVA236 mutation. Preferably the conjugate is characterizedin that said anti-CCR5 antibody of IgG4 subclass has a S228P mutationand said anti-CCR5 antibody of IgG1 subclass has L234A and L235Amutations.

The invention comprises a method for the production of a conjugateaccording to the invention, characterized in that the method comprises

-   a) cultivating a cell containing one or more plasmids containing one    or more nucleic acid molecules encoding a conjugate according to the    invention under conditions suitable for the expression of the    conjugate,-   b) recovering the conjugate from the cell or the supernatant.

In one embodiment are the genes encoding the light and heavy chains ofmAb CCR5 with or without linked antifusogenic peptide located on thesame expression vector or on different expression vectors.

The invention comprises a pharmaceutical composition, containing aconjugate according to the invention, together with a pharmaceuticallyacceptable excipient or carrier.

The invention comprises the use of a conjugate according to theinvention for the manufacture of a medicament for the treatment of viralinfections. Preferably the use is characterized in that the viralinfection is a HIV infection.

The invention comprises the use of a conjugate according to theinvention for the treatment of a patient in need of an antiviraltreatment, preferably an anti HIV treatment.

DESCRIPTION OF THE INVENTION

The current invention reports a conjugate comprising one or moreantifusogenic peptides and an anti-CCR5 antibody (mAb CCR5)characterized in that one to eight antifusogenic peptides are eachconjugated to one terminus of the heavy and/or light chains of saidanti-CCR5 antibody. A number of eight antifusogenic peptides per mAbCCR5 is only possible if the mAb CCR5 comprises eight termini, i.e. iscomposed e.g. of two heavy chains and two light chains. If the mAb CCR5comprises a smaller number of C- and N-termini, e.g. as a scFv, thecorresponding number of antifusogenic peptides possible at maximum inthe conjugate is also reduced, i.e. it is reduced to less than eight.

An “antifusogenic peptide” is a peptide which inhibits events associatedwith membrane fusion or the membrane fusion event itself, including,among other things, the inhibition of infection of uninfected cells by avirus due to membrane fusion. These antifusogenic peptides arepreferably linear peptides. For example, they can be derived from thegp41 ectodomain, e.g. such as DP107, DP178. Examples of such peptidescan be found in U.S. Pat. No. 5,464,933, U.S. Pat. No. 5,656,480, U.S.Pat. No. 6,013,263, U.S. Pat. No. 6,017,536, U.S. Pat. No. 6,020,459,U.S. Pat. No. 6,093,794, U.S. Pat. No. 6,060,065, U.S. Pat. No.6,258,782, U.S. Pat. No. 6,348,568, U.S. Pat. No. 6,479,055, U.S. Pat.No. 6,656,906, WO 1996/19495, WO 1996/40191, WO 1999/59615, WO2000/69902, and WO 2005/067960. For example, the amino acid sequences ofsuch peptides comprise or can be selected from the group of SEQ ID NO: 1to 10 of U.S. Pat. No. 5,464,933; SEQ ID NO:1 to 15 of U.S. Pat. No.5,656,480; SEQ ID NO: 1 to 10 and 16 to 83 of U.S. Pat. No. 6,013,263;SEQ ID NO: 1 to 10, 20 to 83 and 139 to 149 of U.S. Pat. No. 6,017,536;SEQ ID NO:1 to 10, 17 to 83 and 210 to 214 of U.S. Pat. No. 6,093,794;SEQ ID NO:1 to 10, 16 to 83 and 210 to 211 of U.S. Pat. No. 6,060,065;SEQ ID NO:1286 and 1310 of U.S. Pat. No. 6,258,782; SEQ ID NO:1129,1278-1309, 1311 and 1433 of U.S. Pat. No. 6,348,568; SEQ ID NO:1 to 10and 210 to 238 of U.S. Pat. No. 6,479,055; SEQ ID NO:1 to 171, 173 to216, 218 to 219, 222 to 228, 231, 233 to 366, 372 to 398, 400 to 456,458 to 498, 500 to 570, 572 to 620, 622 to 651, 653 to 736, 739 to 785,787 to 811, 813 to 823, 825, 827 to 863, 865 to 875, 877 to 883, 885,887 to 890, 892 to 981, 986 to 999, 1001 to 1003, 1006 to 1018, 1022 to1024, 1026 to 1028, 1030 to 1032, 1037 to 1076, 1078 to 1079, 1082 to1117, 1120 to 1176, 1179 to 1213, 1218 to 1223, 1227 to 1237, 1244 to1245, 1256 to 1268, 1271 to 1275, 1277, 1345 to 1348, 1350 to 1362,1364, 1366, 1368, 1370, 1372, 1374 to 1376, 1378 to 1379, 1381 to 1385,1412 to 1417, 1421 to 1426, 1428 to 1430, 1432, 1439 to 1542, 1670 to1682, 1684 to 1709, 1712 to 1719, 1721 to 1753, 1755 to 1757 of U.S.Pat. No. 6,656,906; or SEQ ID NO:5 to 95 of WO 2005/067960. Theantifusogenic peptide has an amino acid sequence comprising of from 5 to100 amino acids, preferably of from 10 to 75 amino acids and morepreferred of from 15 to 50 amino acids. Especially preferredantifusogenic peptides are C-34, T-20, T-1249, T-651, T-2635, N-36, (M.J. Root et al., Curr. Pharm. Des. (2004) 10:1805-25) and DP-107 (C. Wildet al., Proc. Natl. Acad. Sci. USA (1994) 91:12676-80). One embodimentcomprises the conjugate according to the invention one or moreantifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) wherein i)said antifusogenic peptides are linear peptides with an amino acidsequence of from 5 to 100 amino acids, and ii) one to eightantifusogenic peptides are each conjugated to one terminus of the heavyand/or light chains of said anti-CCR5 antibody. Another embodimentcomprises the conjugate according to the invention having one or moreantifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) wherein i)said antifusogenic peptides are derived from the gp41 ectodomain, andii) one to eight antifusogenic peptides are each conjugated to oneterminus of the heavy and/or light chains of said anti-CCR5 antibody.The term “gp41 ectodomain” denotes the amino acid sequence starting withamino acid position 561 and ending with amino acid position 620 of HIV-1gp 160 or starting with amino acid position 50 and ending with aminoacid position 109 of HIV-1 gp41 (SEQ ID NO:66) (see also e.g. S. Bar andM. J. Alizon, Virol. (2004) 78:811-20).

The term “antibody” encompasses the various forms of antibody structuresincluding whole antibodies and antibody fragments. The antibodyaccording to the invention is preferably a human antibody, a humanizedantibody, a chimeric antibody, a T cell antigen depleted antibody (WO98/33523, WO 98/52976, and WO 00/34317). Genetic engineering ofantibodies is e.g. described in S. L. Morrison et al., Proc. Natl. Acad.Sci. USA (1984) 81:6851-55; U.S. Pat. Nos. 5,202,238 and 5,204,244; L.Riechmann et al., Nature (1988) 332:323-27; M. S, Neuberger et al.,Nature (1985) 314:268-70; N. Lonberg, Nat. Biotechnol. (2005)23:1117-25.

“Antibody fragments” comprise a portion of a full length anti-CCR5antibody, preferably the variable domains thereof or at least theantigen binding portion thereof. Examples of anti-body fragments aree.g. single-chain antibody molecules (scFv), Fab, F(ab)₂ fragments, andthe like as long as they retain the characteristics of an anti-CCR5antibody. ScFv antibodies are, e.g., described in J. S. Huston, Meth.Enzymol. (1991) 203:46-88. Huston also describes linkers and methods forlinking of polypeptides useful for the present invention.

“CCR5” means human CCR5 as described, e.g., in M. Oppermann, CellSignal. (2004) 16:1201-10 and SwissProt P51681. The terms “antibodybinding to CCR5”, “anti-CCR5 antibody”, or “mAb CCR5”, which are usedinterchangeably within this application, mean an antibody specificallybinding to CCR5 and preferably inhibiting HIV fusion with a target cell.Binding can be tested in a cell based in vitro ELISA assay (CCR5expressing CHO cells). Binding is found if the antibody causes an S/N(signal/noise) ratio of 5 or more, preferably 10 or more at an antibodyconcentration of 100 ng/ml. The term “inhibiting HIV fusion with atarget cell” refers to inhibiting HIV fusion with a target cell measuredin an assay comprising contacting said target cell (e.g. PBMC) with thevirus in the presence of the antibody in a concentration effective toinhibit membrane fusion between the virus and said cell and measuringe.g. luciferase reporter gene activity or the HIV p24 antigenconcentration. The term “membrane fusion” refers to fusion between afirst cell coexpressing CCR5 and CD4 polypeptides and a second cell orvirus expressing an HIV env protein. Membrane fusion is determined bygenetically engineered cells and/or viruses by a reporter gene assay(e.g. by luciferase reporter gene assay).

Preferred anti-CCR5 antibodies are mentioned in US 2004-0043033, U.S.Pat. No. 6,610,834, US 2003-0228306, US 2003-0195348, US 2003-0166870,US 2003-0166024, US 2003-0165988, US 2003-0152913, US 2003-0100058, US2003-0099645, US 2003-0049251, US 2003-0044411, US 2003-0003440, U.S.Pat. No. 6,528,625, US 2002-0147147, US 2002-0146415, US 2002-0106374,US 2002-0061834, US 2002-0048786, US 2001/0000241, EP 1 322 332, EP 1263 791, EP 1 207 202, EP 1 161 456, EP 1 144 006, WO 2003/072766, WO2003/066830, WO 2003/033666, WO 2002/083172, WO 02/22077, WO 01/58916,WO 01/58915, WO 01/43779, WO 01/42308, and WO 2006/103100. Especiallypreferred anti-CCR5 antibodies are described in WO 2006/103100. Anespecially preferred anti-CCR5 antibody is characterized in that theantibody comprises a variable heavy chain domain consisting of animmunoglobulin framework and a CDR3 region selected from the groupconsisting of the heavy chain CDR3 sequences SEQ ID NO:16, 17. A furtherpreferred antibody comprises a variable heavy chain region consisting ofan immunoglobulin framework and a CDR3 region selected from the groupconsisting of CDR3 sequences SEQ ID NO: 16, 17, a CDR2 region selectedfrom the group consisting of CDR2 sequences SEQ ID NO:13, 14, 15, and aCDR1 region selected from the group consisting of CDR1 sequences SEQ IDNO:9, 10, 11, 12. Preferred heavy chain variable domains are shown inSEQ ID NO:1, 3, 5, 7. A preferred anti-CCR5 antibody comprises inaddition a variable light chain domain consisting of an immunoglobulinframework and a CDR1 region selected from the group consisting of CDR1sequences SEQ ID NO:18, 19, 20, a CDR2 region selected from the groupconsisting of CDR2 sequences SEQ ID NO:21, 22, 23, and a CDR3 regionselected from the group of CDR3 sequences SEQ ID NO:24, 25. Theanti-CCR5 antibody is preferably characterized in containing as heavychain CDRs the CDRs of SEQ ID NO: 1 and as light chain CDRs the CDRs ofSEQ ID NO:2, as heavy chain CDRs the CDRs of SEQ ID NO:3 and as lightchain CDRs the CDRs of SEQ ID NO:4, as heavy chain CDRs the CDRs of SEQID NO:5 and as light chain CDRs the CDRs of SEQ ID NO:6, or as heavychain CDRs the CDRs of SEQ ID NO:7 and as light chain CDRs the CDRs ofSEQ ID NO:8.

CDR sequences can be determined according to the standard definition ofE. A. Kabat et al., Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, National Institutes of Health, Bethesda, Md.(1991). CDRs of SEQ ID NO: 1-8 are shown in SEQ ID NO:9-25.

The anti-CCR5 antibody comprises preferably a variable heavy and lightchain domain independently selected from the group consisting of

-   a) the heavy chain (V_(H)) variable domain defined by amino acid    sequence SEQ ID NO: 1 and the light chain (V_(L)) variable domain    defined by SEQ ID NO:2;-   b) the heavy chain variable domain defined by amino acid sequence    SEQ ID NO:3 and the light chain variable domain defined by SEQ ID    NO:4;-   c) the heavy chain variable domain defined by amino acid sequence    SEQ ID NO:5 and the light chain variable domain defined by SEQ ID    NO:6;-   d) the heavy chain variable domain defined by amino acid sequence    SEQ ID NO:7 and the light chain variable domain defined by SEQ ID    NO:8.

The antibody used in the conjugate according to the invention ispreferably characterized in that the constant domains are of humanorigin. Such constant domains are well known in the state of the artand, e.g., described by Kabat (see e.g. G. Johnson and T. T. Wu, NucleicAcids Res. (2000) 28:214-18). For example, a useful human IgG1 heavychain constant region (C_(H)1-Hinge-C_(H)2-C_(H)3) comprises an aminoacid sequence independently selected from the group consisting of SEQ IDNO:26, 27. For example, a useful human kappa (κ) light chain constantdomain comprises an amino acid sequence of a kappa light chain constantdomain (κ light chain constant domain, C_(L)) of SEQ ID NO:28. It isfurther preferred that the antibody's variable domains are of mouseorigin and comprises the antibody variable domain sequence frame of amouse antibody according to Kabat (see e.g. G. Johnson and T. T. Wu,supra).

A preferred anti-CCR5 antibody shows a binding to the same epitope(s) ofCCR5 as does an antibody selected from the group consisting of theantibodies A to E or is inhibited in binding to CCR5 by antibodies A toE due to steric hindrance of binding or competitive binding. Epitopebinding is investigated by using alanine scanning according to themethod described by W. C. Olson et al. (J. Virol. (1999) 73:4145-55) forepitope mapping. A signal reduction of 75% or more shows that themutated amino acid(s) contribute to the epitope recognized by saidantibody. Binding of the antibody to the same epitope is found, if theamino acids contributing to the epitope are recognized by theinvestigated antibody and antibody A, B, C, D, or E. Antibody C, whichshows lower IC₅₀ values than antibody 2D7 in HIV assays, binds to anepitope including amino acids on the ECL2 domain of CCR5 (B. Lee et al.,J. Biol. Chem. (1999) 274:9617-26) which is different from the epitoperecognized by antibody 2D7 (2D7 binds to amino acids K171 and E172 ofECL2A but not to ECL2B amino acids 184-189). Epitope binding forantibody C is found to be 20% for CCR5 mutant K171A or E172A (glu 172 ismutated to ala). 100% epitope binding is defined for wild-type CCR5. Afurther preferred anti-CCR5 antibody binds to the same epitope asantibody C binds.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. Preferably an antibody according to the invention bindsspecifically to native but not to denatured CCR5. Such an antibodycomprises preferably heavy chain CDR3 of SEQ ID NO: 17, and preferablyin addition heavy chain CDRs selected from the group of CDRs of SEQ IDNO:10, 11, 12, 14 and/or 15. Preferably such an antibody is antibody B,C, D, or E, or comprises the variable domains of antibody B, C, D, or E.Preferably an antibody binding to denatured CCR5 is antibody A orcomprises the variable domains of antibody A.

The term “variable domain” (variable domain of a light chain (V_(L)),variable domain of a heavy chain (V_(H))) as used herein denotes eachdomain of the pair of light and heavy chain domains which is involveddirectly in the binding of the antibody to the antigen. The variabledomains of the light and heavy chain have the same general structure,i.e. they possess an “immunoglobulin framework”, and each domaincomprises four “framework regions” (FR), whose sequences are widelyconserved, connected by three “hypervariable regions” (or“complementarity determining regions”, CDRs). The framework regionsadopt a β-sheet conformation and the CDRs may form loops connecting theβ-sheet structure. The CDRs in each chain are held in theirthree-dimensional structure by the framework regions and form togetherwith the CDRs from the other chain the antigen binding site. Theantibody heavy and light chain CDR3 regions play a particularlyimportant role in the binding specificity/affinity of the antibodiesaccording to the invention and therefore provide a further object of theinvention.

The terms “antigen-binding portion of an antibody” or “antigen-bindingsite of an antibody” when used herein refer to the amino acid residuesof an antibody which are responsible for antigen-binding. Theantigen-binding site of an antibody comprises amino acid residues fromthe “complementarity determining regions” or “CDRs”. “Framework” or “FR”regions are those variable domain regions other than the hypervariableregion residues as herein defined. Therefore, the light and heavy chainvariable domains of an antibody comprise from N- to C-terminus theregions FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 (immunoglobulinframework). Especially, the CDR3 region of the heavy chain is the regionwhich contributes most to antigen binding and defines the antibody.Preferably the antibody according to the invention is characterized bycomprising in its heavy chain variable domain the CDR3 sequence of SEQID NO:16 or SEQ ID NO:17. Complementarity determining (CDR) andframework (FR) regions are determined according to the standarddefinition of E. A. Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991).

The “Fc part” of an anti-CCR5 antibody is not involved directly inbinding to CCR5, but exhibit various effector functions. Depending onthe amino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2.According to the heavy chain constant regions the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Theantibodies according to the invention are preferably of IgG type. An “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on basis of papain cleavage of antibodies. The antibodiesaccording to the invention contain as Fc part a human Fc part or an Fcpart derived from human origin. In a further embodiment of the inventionthe Fc part is either an Fc part of a human antibody of the subclassIgG4 or an Fc part of a human antibody of the subclass IgG1, IgG2, orIgG3, which is modified in such a way that no Fcγ receptor (e.g.FcγRIIIa) binding and/or no C1q binding as defined below can bedetected. Preferably the Fc part is a human Fc part and especiallypreferred either from human IgG4 subclass or a mutated Fc part fromhuman IgG1 subclass. Further preferred are Fc parts from human IgG1subclass with mutations L234A and L235A. Further preferred are Fc partsshown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 26 with mutationsL234A and L235A, SEQ ID NO: 27 with mutation S228P. While IgG4 showsreduced Fcγ receptor (FcγRIIIa) binding, antibodies of other IgGsubclasses show strong binding. However Pro238, Asp265, Asp270, Asn297(loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237,Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residueswhich if altered provide also reduced Fcγ receptor binding (R. L.Shields et al., J. Biol. Chem. (2001) 276:6591-604; J. Lund et al.,FASEB J. (1995) 9:115-19; A. Morgan et al., Immunol. (1995) 86:319-24;EP 0 307 434). Preferably an antibody according to the invention is inregard to Fcγ receptor binding of IgG4 subclass or of IgG1 or IgG2subclass, with a mutation in L234, L235, and/or D265, and/or containsthe PVA236 mutation. Preferred are the mutations S228P, L234A, L235A,L235E, and/or PVA236 (PVA236 means that the amino acid sequence ELLG(given in one letter amino acid code) from amino acid position 233 to236 of IgG1 or EFLG of IgG4 is replaced by PVA). Especially preferredare the mutations S228P of IgG4, and L234A and L235A of IgG1. The Fcpart of an antibody is directly involved in ADCC (antibody-dependentcell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity).Complement activation (CDC) is initiated by binding of complement factorC1q to the Fc part of most IgG antibody subclasses. Binding of C1q to anantibody is caused by defined protein-protein interactions at the socalled binding site. Such Fc part binding sites are known in the stateof the art and described e.g. by T. J. Lukas et al., J. Immunol. (1981)127:2555-60; R. Brunhouse and J. J. Cebra, Mol. Immunol. (1979) 16:907-17; D. R. Burton et al., Nature (1980) 288:338-44; J. E. Thommesenet al., Mol. Immunol. (2000) 37:995-1004; E. E. Idusogie et al., J.Immunol. (2000) 164:4178-84; M. Hezareh et al., J. Virol. (2001)75:12161-68; A. Morgan et al., Immunol. (1995) 86:319-24; and EP 0 307434. Such Fc part binding sites are, e.g., characterized by the aminoacids L234, L235, D270, N297, E318, K320, K322, P331, and P329(numbering according to EU index of Kabat). Antibodies of subclass IgG1,IgG2, and IgG3 usually show complement activation including C1q and C3binding, whereas IgG4 does not activate the complement system and doesnot bind C1q and C3. An anti-CCR5 antibody which does not bind Fcγreceptor and/or complement factor C1q does not elicit antibody-dependentcellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity(CDC). Preferably, this antibody is characterized in that it binds CCR5,contains an Fc part derived from human origin, and does not bind Fcγreceptors and/or complement factor C1q. More preferably, this antibodyis a human, or humanized, or a T-cell antigen depleted antibody. C1qbinding can be measured according to Idusogie, E. E., et al., J.Immunol. 164 (2000) 4178-4184. No “C1q binding” is found if in such anassay the optical density (OD) at 492-405 nm is for the test antibodylower than 15% of the value for human C1q binding of the unmodifiedwild-type antibody Fc part at an antibody concentration of 8 μg/ml. ADCCcan be measured as binding of the antibody to human FcγRIIIa on human NKcells. Binding is determined at an antibody concentration of 20 μg/ml.“No Fcγ receptor binding” or “no ADCC” means a binding of up to 30% tohuman FcγRIIIa on human NK cells at an antibody concentration of 20μg/ml compared to the binding of the same antibody as human IgG1 (SEQ IDNO:26).

An antibody used in a conjugate according to the invention include, inaddition, such antibodies having “conservative sequence modifications”(variant antibodies), which are amino acid sequence modifications whichdo not affect or alter the above-mentioned characteristics of theantibody according to the invention. Modifications can be introduced bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative amino acid substitutionsinclude ones in which the amino acid residue is replaced with an aminoacid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g. lysine,arginine, histidine), acidic side chains (e.g. aspartic acid, glutamicacid), uncharged polar side chains (e.g. glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), non-polar sidechains (e.g. alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g. threonine,valine, isoleucine), and aromatic side chains (e.g. tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a human anti-CCR5 antibody can be preferablyreplaced with another amino acid residue from the same side chainfamily. A “variant” anti-CCR5 antibody, refers therefore herein to amolecule which differs in amino acid sequence from a “parent” anti-CCR5antibody's amino acid sequence by up to ten, preferably from about twoto about five, additions, deletions, and/or substitutions in one or moreof the variable domain regions of the parent antibody outside the heavychain CDR3 region. Each other heavy chain CDR region comprises atmaximum one single amino acid addition, deletion, and/or substitution.The invention comprises a method of modifying the CDR amino acidsequence of a parent antibody binding to CCR5, characterized inselecting a heavy chain variable domain from the group of heavy chainvariable domains consisting of SEQ ID NO: 1, 3, 5, 7, and/or a lightchain variable domain from the group of light chain variable domainsconsisting of SEQ ID NO:2, 4, 6, 8, providing a nucleic acid encodingsaid initial variable domain amino acid sequence, modifying said nucleicacid in that one amino acid is modified in heavy chain CDR1, one aminoacid is modified in heavy chain CDR2, 1-3 amino acid are modified inlight chain CDR1, 1-3 amino acids are modified in light chain CDR2,and/or 1-3 amino acids are modified in light chain CDR3, expressing andincorporating said modified variable domain(s) amino acid sequence in anantibody structure, measuring whether said antibody binds to CCR5 andselecting said modified variable domain(s)/CDR(s) if the antibody bindsto CCR5. Preferably such modifications are conservative sequencemodifications. Amino acid sequence modifications can be performed bymutagenesis based on molecular modeling as described by L. Riechmann etal., Nature (1988) 332:323-27, and C. Queen et al., Proc. Natl. Acad.Sci. USA (1989) 86:10029-33.

The term “linker” or “peptidic linker” as used within this applicationdenotes peptide linkers of natural and/or synthetic origin. They arebuilding up of a linear amino acid chain wherein the 20 naturallyoccurring amino acids are the monomeric building blocks. The chain has alength of from 1 to 50 amino acids, preferred between 1 and 28 aminoacids, especially preferred between 3 and 25 amino acids. The linker maycontain repetitive amino acid sequences or sequences of naturallyoccurring polypeptides, such as polypeptides with a hinge-function. Thelinker has the function to ensure that a peptide conjugated to ananti-CCR5 antibody can perform its biological activity by allowing thepeptide to fold correctly and to be presented properly. Preferably thelinker is a “synthetic peptidic linker” that is designated to be rich inglycine, glutamine, and/or serine residues. These residues are arrangede.g. in small repetitive units of up to five amino acids, such as GGGGS,QQQQG, or SSSSG. This small repetitive unit may be repeated for two tofive times to form a multimeric unit. At the amino- and/orcarboxy-terminal ends of the multimeric unit up to six additionalarbitrary, naturally occurring amino acids may be added. Other syntheticpeptidic linkers are composed of a single amino acid, that is repeatedbetween 10 to 20 times, such as e.g. serine in the linkerSSSSSSSSSSSSSSS. At each of the amino- and/or carboxy-terminal end up tosix additional arbitrary, naturally occurring amino acids may bepresent. Preferred linkers are shown in Table 2. Especially preferredare linkers [GQ₄]₃GNN (SEQ ID NO:40), LSLSPGK (SEQ ID NO:36), LSPNRGEC(SEQ ID NO:37), LSLSGG (SEQ ID NO:61), LSLSPGG (SEQ ID NO:62). Allpeptidic linkers can be encoded by a nucleic acid molecule and thereforecan be recombinantly expressed. As the linkers are themselves peptides,the antifusogenic peptide is connected to the linker via a peptide bondthat is formed between two amino acids. The peptidic linker isintroduced between the antifusogenic peptide and the anti-CCR5 antibodychain to which the antifusogenic peptide is to be conjugated. Thereforetwo or three, respectively, possible sequences (in amino- tocarboxy-terminal direction) exist: a) antifusogenic peptide-peptidiclinker-anti-CCR5 antibody polypeptide chain, or b) anti-CCR5 antibodypolypeptide chain-peptidic linker-antifusogenic peptide, or c)antifusogenic peptide-peptidic linker-anti-CCR5 antibody polypeptidechain-peptidic linker-antifusogenic peptide.

In one embodiment of the invention the conjugate is characterized incomprising i) the heavy chain (V_(H)) variable domain defined by aminoacid sequence SEQ ID NO:1 and the light chain (V_(L)) variable domaindefined by SEQ ID NO:2; or the heavy chain variable domain defined byamino acid sequence SEQ ID NO:3 and the light chain variable domaindefined by SEQ ID NO:4; or the heavy chain variable domain defined byamino acid sequence SEQ ID NO:5 and the light chain variable domaindefined by SEQ ID NO:6; or the heavy chain variable domain defined byamino acid sequence SEQ ID NO:7 and the light chain variable domaindefined by SEQ ID NO:8; ii) a linker selected from the group consistingof the amino acids glycine (G) and asparagine (N), the tripeptide GST,and SEQ ID NO:36-62; and iii) an antifusogenic peptide selected from thegroup of peptides defined by SEQ ID NO:29 to 35.

A preferred conjugate of a heavy and/or light chain of mAb CCR5 and anantifusogenic peptide(s) (“chain conjugate”) is selected from the groupconsisting of the conjugates (1) [antifusogenicpeptide]-[linker]_(m)-[heavy chain], (2) [heavychain]-[linker]_(m)-[antifusogenic peptide], (3) [antifusogenicpeptide]-[linker]_(m)-[heavy chain]-[antifusogenic peptide], (4)[antifusogenic peptide]-[linker]_(m)-[light chain], (5) [lightchain]-[linker]_(m)-[antifusogenic peptide], (6) [antifusogenicpeptide]-[linker]_(m)-[light chain]-[antifusogenic peptide], (7)[antifusogenic peptide]-[linker]_(m)-[heavychain]-[linker]_(m)-[antifusogenic peptide], (8) [antifusogenicpeptide]-[linker]_(m)-[light chain]-[linker]_(m)-[antifusogenicpeptide], wherein the linker can be the same or different both withinand between said chain conjugates, wherein m is an integer of 1 or 0,and m can be independently the same or different both within and betweensaid conjugates. For example in a conjugate comprising a chain conjugate(7) and a mAb CCR5 light chain the two linkers in chain conjugate (7)can be the same, i.e. have the same amino acid sequence and length, orcan be different, i.e. have different amino acid sequences and/orlengths, or one or both can be absent. For example in a conjugatecomprising chain conjugates (2) and (4) the linker contained in chainconjugate (2) and the linker contained in chain conjugate (4) can be thesame, i.e. have the same amino acid sequence and length, or can bedifferent, i.e. have different amino acid sequences and/or lengths, orone or both can be absent. In the chain conjugates the linker(s) can bepresent (m=1) or absent (m=0). Preferred chain conjugates are the chainconjugates (2), (3), (4), and (7). One embodiment of the currentinvention is a conjugate comprising 2×[mAb CCR5 light chain] and 2×chain conjugate (2). This conjugate comprises two not conjugateanti-CCR5 antibody light chains and two anti-CCR5 antibody heavy chainsconjugated via the C-terminus to the N-terminus of an antifusogenicpeptide, optionally with an intermediate linker. Another embodiment ofthe current invention is a conjugate comprising two mAb CCR5 lightchains and two chain conjugates (3). Still another embodiment is aconjugate comprising two mAb CCR5 heavy chains and two chain conjugates(4). A further embodiment of the current invention is a conjugatecomprising two mAb CCR5 light chains and two chain conjugates (7). Theheavy and/or light chain comprises preferably a constant region (Fc).

The invention further provides a method for the manufacture of apharmaceutical composition comprising an effective amount of a conjugateaccording to the invention together with a pharmaceutically acceptablecarrier and the use of the conjugate according to the invention for sucha method.

The invention further provides the use of a conjugate according to theinvention in an effective amount for the manufacture of a pharmaceuticalagent, preferably together with a pharmaceutically acceptable carrier,for the treatment of a patient suffering from AIDS.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

Methods and techniques known to a person skilled in the art, which areuseful for carrying out the current invention, are described e.g. in F.M. Ausubel, ed., Current Protocols in Molecular Biology, Volumes I toIII (1997), Wiley and Sons; Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989).

In the conjugates according to the invention the carboxy-terminal aminoacid of an anti-CCR5 antibody chain is conjugated via a peptide bond tothe amino-terminal amino acid of the antifusogenic peptide or thecarboxy-terminal amino acid of the antifusogenic peptide is conjugatedvia a peptide bond to the amino-terminal amino acid of an anti-CCR5antibody chain. In one embodiment an intermediate linker is presentbetween the antifusogenic peptide and the anti-CCR5 antibody chain.Thus, the conjugate according to the invention is characterized by thegeneral formula

mAb CCR5-[linker]_(m)-[antifusogenic peptide]_(n)

wherein m is independently for each antifusogenic peptide either 0 (i.e.a direct peptide bond between mAb CCR5 and the antifusogenic peptide) or1 (i.e. a linker is present between mAb CCR5 and antifusogenic peptide)and n is an integer of from 1 to 8. In one embodiment n is an integer offrom 2 to 8. In another embodiment n is an integer of from 2 to 4. Inanother embodiment n is an integer of 2 or 4. One embodiment of theinvention comprises a conjugate characterized in comprising anantifusogenic peptide at each C-terminus of the heavy chains or at eachN-terminus of the light chains of the anti-CCR5 antibody. In thisembodiment two antifusogenic peptides are conjugated to one anti-CCR5antibody. In an other embodiment is the conjugate characterized bycomprising an antifusogenic peptide at each C-terminus of the heavychains and at each N-terminus of the light chains. In this embodimentfour antifusogenic peptides are conjugated to one anti-CCR5 antibody.

The antifusogenic peptide which is introduced at a terminus of a mAbCCR5 heavy and/or light chain(s) is small of size compared to the mAbCCR5. For example, the smallest immunoglobulins, immunoglobulins ofclass G, have a molecular weight of approximately 150 kDa; anantifusogenic peptide has preferably a size (molecular weight) of lessthan 12.5 kDa, which is equivalent to about 100 amino acids, in generalless than 7.5 kDa, which is equivalent to about 60 amino acids. Theantifusogenic peptide has an amino acid sequence of from 5 to 100 aminoacid residues, preferably of from 10 to 75 amino acid residues, morepreferably of from 15 to 50 amino acid residues. The conjugates of thecurrent invention are useful for pharmaceutical, therapeutical, ordiagnostical applications. The number of antifusogenic peptides, whichcan be conjugated to mAb CCR5 heavy and/or light chain(s), is from oneto the combined number of amino- and carboxy-termini of the anti-CCR5antibody polypeptide chains. As the current invention encompassesdifferent anti-CCR5 antibodies the number of antifusogenic peptides canvary. In case of an anti-CCR5 antibody comprising two heavy and twolight chains the combined number of amino-termini (N-termini) andcarboxy-termini (C-termini) is eight, which is at the same time thetotaling maximum number of conjugated antifusogenic peptides; in casee.g. of an anti-CCR5 antibody fragment such as a single chain antibody(scFv) the combined number of termini and therefore the maximum numberof conjugatable antifusogenic peptides is two. If a single antifusogenicpeptide is conjugated to mAb CCR5, the peptide can occupy any one of thetermini of the anti-CCR5 antibody chains. Likewise, if the maximumpossible number of peptides is conjugated to mAb CCR5, all termini areoccupied by a single peptide. If the number of peptides, which areconjugated to mAb CCR5, is smaller than the maximum possible number,different distributions of the peptides at the termini of the anti-CCR5antibody chains are possible. For example, if four peptides areconjugated to an immunoglobulin of the G or E class, five differentcombinations are possible (see Table 1). In two combinations all terminiof one kind, i.e. all four amino-termini or all four carboxy-termini ofthe anti-CCR5 antibody chains, are each conjugated to one singleantifusogenic peptide. The other termini are not conjugated. Thisresults in one embodiment in an allocation of themodifications/conjugations in one area of the anti-CCR5 antibody. In theother cases the polypeptides are conjugated to a number of both termini.Within these combinations the conjugated peptides are allocated todifferent areas of the anti-CCR5 antibody. In either case the sum ofconjugated termini is four.

TABLE 1 Possible combination for the conjugation of four peptides to thetermini of an anti-CCR5 antibody composed of four polypeptide chains.number of occupied number of occupied total number of amino-terminicarboxy-termini occupied termini 4 0 4 3 1 4 2 2 4 1 3 4 0 4 4

The current invention preferably comprises conjugates in which at leasttwo of the termini are conjugated to an antifusogenic peptide. The aminoacid sequences of the antifusogenic peptides can be different, similaror identical. In one embodiment the amino acid sequence identity is inthe range of from 90% to less than 100%; these amino acid sequences andthe corresponding peptides are defined as similar. In a preferredembodiment the antifusogenic peptides are identical, i.e. have an aminoacid identity of 100%.

The present invention comprises a conjugate comprising one or moreantifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) wherein oneto eight antifusogenic peptides are each conjugated to one terminus ofthe heavy and/or light chains of said anti-CCR5 antibody via a peptidebond. In one embodiment the conjugate according to the inventioncomprises at least two antifusogenic peptides and an anti-CCR5 antibodywherein two to eight antifusogenic peptides are each conjugated to oneterminus of the heavy and/or light chains of said anti-CCR5 antibody.

In one embodiment the conjugate according to the invention ischaracterized i) in comprising two light chain variable domains of SEQID NO:2, two chain conjugates of type (2) each comprising a heavy chainvariable domain of SEQ ID NO: 1, a linker of SEQ ID NO:40 and anantifusogenic peptide of SEQ ID NO:33, ii) in comprising two light chainvariable domains of SEQ ID NO:4, two chain conjugates of type (2) eachcomprising a heavy chain variable domain of SEQ ID NO:3, a linker of SEQID NO:40 and an antifusogenic peptide of SEQ ID NO:33, iii) incomprising two light chain variable domains of SEQ ID NO:6, two chainconjugates of type (2) each comprising a heavy chain variable domain ofSEQ ID NO:5, a linker of SEQ ID NO:40 and an antifusogenic peptide ofSEQ ID NO:33, or iv) in comprising two light chain variable domains ofSEQ ID NO:8, two chain conjugates of type (2) each comprising a heavychain variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and anantifusogenic peptide of SEQ ID NO:33.

The conjugation between the antifusogenic peptide and the anti-CCR5antibody is performed on the nucleic acid level. Therefore a peptidebond is formed between the antifusogenic peptide and the anti-CCR5antibody chain with or without an intermediate linker. Thus either thecarboxy-terminal amino acid of the antifusogenic peptide is conjugatedto the amino-terminal amino acid of an anti-CCR5 antibody chain with orwithout an intermediate linker, or a carboxy-terminal amino acid of theanti-CCR5 antibody chain is conjugated to the amino-terminal amino acidof the antifusogenic peptide with or without an intermediate linker orboth termini of the anti-CCR5 antibody chain are conjugated to anantifusogenic peptide each with or without an intermediate linker. Forthe recombinant production of the antifusogenic peptide-anti-CCR5antibody-conjugate according to the invention one or more nucleic acidmolecules encoding different polypeptides are required, preferably twoto eight nucleic acid molecules are employed. These nucleic acidmolecules encode the different anti-CCR5 antibody polypeptide chains ofthe conjugate and are in the following referred to as structural genes.They can be located on the same expression plasmid (vector) or canalternatively be located on different expression plasmids (vectors). Theassembly of the conjugate takes preferably place before secretion of theconjugate and thus within the expressing cells. Therefore the nucleicacid molecules encoding the polypeptide chains of the conjugate arepreferably expressed in the same host cell. If after recombinantexpression a mixture of conjugates is obtained, the conjugates can beseparated and purified by methods known to a person skilled in the art.These methods are well established and widespread used forimmunoglobulin purification and are employed either alone or incombination. Such methods are, for example, affinity chromatographyusing microbial-derived proteins (e.g. protein A or protein G affinitychromatography), ion exchange chromatography (e.g. cation exchange(carboxymethyl resins), anion exchange (amino ethyl resins) andmixed-mode exchange chromatography), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and preparativeelectrophoretic methods (such as gel electrophoresis, capillaryelectrophoresis) (M. A. Vijayalakshmi, Appl. Biochem. Biotech. (1998)75:93-102). With recombinant engineering methods known to a personskilled in the art the conjugates can be tailor-made on the nucleicacid/gene level. The nucleic acid sequences encoding immuno-globulinsare known and can be obtained for example from genomic databases.Likewise the nucleic acid sequences encoding antifusogenic peptides areknown or can easily be deduced from their amino acid sequence. Theelements required for the construction of an expression plasmid for theexpression of the conjugate of the current invention are, for example,an expression cassette for the anti-CCR5 antibody light chain in itsnatural and/or modified and/or conjugated version, an expressioncassette for the anti-CCR5 antibody heavy chain in its natural and/ormodified and/or conjugated version (alternatively the anti-CCR5 antibodylight chain and the anti-CCR5 antibody heavy chain can be contained inthe same expression cassette, e.g. as bicistronic expression element), aselection marker, and an E. coli replication as well as selection unit.These expression cassettes comprise a promoter, a DNA segment encoding asecretion signal sequence, the structural gene, and aterminator/polyadenylation signal. The elements are assembled in anoperatively linked form either on one plasmid encoding all chains of theconjugate, or on two or more plasmids each encoding one or more chainsof the conjugate. For the expression of the encoded polypeptides theplasmid(s) is (are) introduced into a suitable host cell. Proteins arepreferably produced in mammalian cells such as CHO cells, NS0 cells,Sp2/0 cells, COS cells, HEK cells, K562 cells, BHK cells, PER.C6® cells,and the like. The regulatory elements of the plasmid have to be selectedin a way that they are functional in the selected host cell. For theexpression the host cell containing the plasmid encoding one or morechains of the conjugate is cultivated under conditions suitable for theexpression of the chains. The expressed conjugate chains arefunctionally assembled. The fully processed antifusogenicpeptide-anti-CCR5 antibody-conjugate is secreted into the medium.

An “expression plasmid” is a nucleic acid encoding a polypeptide to beexpressed in a host cell. Typically, an expression plasmid comprises aprokaryotic plasmid propagation unit, e.g. for E. coli, comprising anorigin of replication, and a resistance gene, an eukaryotic selectionmarker, and one or more expression cassettes for the expression of thestructural gene(s) of interest comprising a promoter, a structural gene,and a transcription terminator including a polyadenylation signal. Geneexpression is usually placed under the control of a promoter, and such astructural gene is said to be “operably linked to” the promoter.Similarly, a regulatory element and a core promoter are operably linkedif the regulatory element modulates the activity of the core promoter.

One aspect of the current invention is thus a method for the productionof a conjugate according to the invention, comprising the followingsteps

-   a) cultivating a cell containing one or more expression plasmids    each comprising one or more nucleic acid molecules encoding a    conjugate according to the invention under conditions suitable for    the expression of the conjugate,-   b) recovering the conjugate from the cell or the supernatant.

The term “under conditions suitable for the expression of the conjugate”denotes conditions which are used for the cultivation of a cellexpressing a polypeptide and which are known to or can easily bedetermined by a person skilled in the art. It is known to a personskilled in the art that these conditions may vary depending on the typeof cell cultivated and type of polypeptide expressed. In general thecell is cultivated at a temperature, e.g. between 20° C. and 40° C., andfor a period of time sufficient to allow effective production of thepolypeptide conjugate, e.g. for 4 to 28 days.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption/resorption delaying agents, and the likethat are physiologically compatible. Preferably, the carrier is suitablefor injection or infusion. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for thepreparation of sterile injectable solutions or dispersion. The use ofsuch media and agents for pharmaceutically active substances is known inthe art. In addition to water, the carrier can be, for example, anisotonic buffered saline solution.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient, which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, other drugs, compounds and/or materials used in combinationwith the particular compositions employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

The invention preferably comprises the use of a conjugate according tothe invention for the treatment of a patient suffering fromimmunodeficiency syndromes such as AIDS.

The following examples, sequence listing, figures and deposits areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: Plasmid map of mab CCR5 κ-light chain expression vector 4900.

FIG. 2: Plasmid map of mAb CCR5 γ1-heavy chain expression vector 4901.

FIG. 3: Plasmid map of mAb CCR5 γ1-heavy chain conjugate expressionvector 4995.

Anti-CCR5 Antibody Deposition

Preferred hybridoma cell lines expressing mAb CCR5 useful in theconjugates according to the invention were deposited with DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany.

Cell line Deposition No. Date of Deposit m<CCR5>Pz01.F3 DSM ACC 2681 18Aug. 2004 m<CCR5>Pz02.1C11 DSM ACC 2682 18 Aug. 2004 m<CCR5>Pz03.1C5 DSMACC 2683 18 Aug. 2004 m<CCR5>Pz04.1F6 DSM ACC 2684 18 Aug. 2004

Antibody Nomenclature

<CCR5>Pz01.F3: Antibody A SEQ ID NO: 1, 2 <CCR5>Pz02.1C11: Antibody BSEQ ID NO: 3, 4 <CCR5>Pz03.1C5: Antibody C SEQ ID NO: 5, 6<CCR5>F3.1H12.2E5: Antibody D SEQ ID NO: 7, 8 <CCR5>Pz04.1F6: Antibody E

CCR 5 Antibody Sequences, Sequences of Antifusogenic Peptides andSequences of Peptidic Linkers

SEQ ID NO: 1 <CCR5>Pz01.F3 heavy chain, variable domain SEQ ID NO: 2<CCR5>Pz01.F3 light chain, variable domain SEQ ID NO: 3 <CCR5>Pz02.1C11heavy chain, variable domain SEQ ID NO: 4 <CCR5>Pz02.1C11 light chain,variable domain SEQ ID NO: 5 <CCR5>Pz03.1C5 heavy chain, variable domainSEQ ID NO: 6 <CCR5>Pz03.1C5 light chain, variable domain SEQ ID NO: 7<CCR5>F3.1H12.2E5 heavy chain, variable domain SEQ ID NO: 8<CCR5>F3.1H12.2E5 light chain, variable domain SEQ ID NO: 9 Heavy chainCDR1 SEQ ID NO: 10 Heavy chain CDR1 SEQ ID NO: 11 Heavy chain CDR1 SEQID NO: 12 Heavy chain CDR1 SEQ ID NO: 13 Heavy chain CDR2 SEQ ID NO: 14Heavy chain CDR2 SEQ ID NO: 15 Heavy chain CDR2 SEQ ID NO: 16 Heavychain CDR3 SEQ ID NO: 17 Heavy chain CDR3 SEQ ID NO: 18 Light chain CDR1SEQ ID NO: 19 Light chain CDR1 SEQ ID NO: 20 Light chain CDR1 SEQ ID NO:21 Light chain CDR2 SEQ ID NO: 22 Light chain CDR2 SEQ ID NO: 23 Lightchain CDR2 SEQ ID NO: 24 Light chain CDR3 SEQ ID NO: 25 Light chain CDR3SEQ ID NO: 26 γ1 heavy chain constant region SEQ ID NO: 27 γ4 heavychain constant region SEQ ID NO: 28 κ light chain constant domain SEQ IDNO: 29 C34 SEQ ID NO: 30 T20 SEQ ID NO: 31 T1249 SEQ ID NO: 32 T651 SEQID NO: 33 T2635 SEQ ID NO: 34 N36 SEQ ID NO: 35 DP107 SEQ ID NO: 36-62linker peptides SEQ ID NO: 63 Amino acid sequence of mature mAb CCR5κ-light chain SEQ ID NO: 64 Amino acid sequence of mature mAb CCR5γ1-heavy chain SEQ ID NO: 65 Amino acid sequence of mature mAb CCR5conjugate heavy chain SEQ ID NO: 66 HIV-1 gp41

TABLE 2 Linker No. Linker peptides SEQ ID NO: 1 LSLSPGK 36 2 LSPNRGEC 373 [GQ₄]₃ 38 4 [GQ₄]₃G 39 5 [GQ₄]₃GNN 40 6 GGG[SG₄]₂SGG 41 7 GGG[SG₄]₂SGN42 8 [SG₄]₃ 43 9 [SG₄]₃G 44 10 G[SG₄]₃T 45 11 [SG₄]₃GG 46 12 [SG₄]₃GGT47 13 [SG₄]₃GGN 48 14 [SG₄]₃GAS 49 15 [SG₄]₅ 50 16 [SG₄]₅G 51 17[SG₄]₅GG 52 18 [SG₄]₅GAS 53 19 G(S)₁₅G 54 20 G(S)₁₅GAS 55 21 G — 22 N —23 GST — 24 [(G)₄S]₃GAS 56 25 [(G)₄S]₃G 57 26 [(G)₄S]₅G 58 27 [(G)₄S]₃GG59 28 [(G)₄S]₅GG 60 29 LSLSGG 61 30 LSLSPGG 62

EXAMPLES Materials & Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: E. A. Kabat et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Aminoacids of antibody chains are numbered according to EU numbering (G. M.Edelman et al., Proc. Natl. Acad. Sci. USA (1969) 63: 78-85; E. A. Kabatet al., supra).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in J. Sambrooket al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 100-600 bp long gene segments, which are flankedby singular restriction endonuclease cleavage sites, were assembled byannealing and ligation of oligonucleotides including PCR amplificationand subsequently cloned into the pCR2.1-TOPO-TA cloning vector(Invitrogen Corp., USA) via A-overhangs. The DNA sequence of thesubcloned gene fragments were confirmed by DNA sequencing.

Protein Determination

The protein concentration of the conjugate was determined by determiningthe optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Example 1 Synthesizing the Expression Plasmids

The gene segments encoding an anti-CCR5 antibody light chain variabledomain (V_(L)) and the human kappa-light chain constant domain (C_(L))were joined as were gene segments for the anti-CCR5 antibody heavy chainvariable domain (V_(H)) and the human γ1-heavy chain constant domains(C_(H)1-Hinge-C_(H)2-C_(H)3).

In the case of mAb CCR5 of SEQ ID NO:63/64 the heavy and light chainvariable domains are derived from a mouse antibody and the heavy andlight chain constant domains are derived from a human antibody (C-kappaand IgG1).

Subsequently, the gene segment encoding a complete anti-CCR5 antibodylight chain was joined at the N- and/or C-terminus with a nucleic acidencoding an antifusogenic peptide including a connecting linker sequenceand/or the gene segment encoding a complete anti-CCR5 antibody heavychain was joined at the N- and/or C-terminus with a nucleic acidencoding an antifusogenic peptide including a connecting linkersequence.

a) Vector 4900

Vector 4900 is an expression plasmid for transient expression of a mAbCCR5 light chain (genomically organized expression cassette; exon-intronorganization) in HEK293 cells.

Beside the mAb CCR5 κ-light chain expression cassette this vectorcontains:

-   -   a hygromycine resistance gene as a selectable marker,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the mAb CCR5 κ-light chain gene is composed ofthe following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   a synthetic 5′-untranslated region,    -   a murine immunoglobulin heavy chain signal sequence including a        signal sequence intron (signal sequence 1, intron, signal        sequence 2 [L1-intron-L2]),    -   the murine anti-CCR5 antibody mature variable κ-light chain        encoding segment arranged with a unique BsmI restriction site at        the 5′-end (L2 signal sequence) and a splice donor site and a        unique NotI restriction site at the 3′-end,    -   a human/mouse κ-light chain hybrid intron 2,    -   the human κ-light gene constant domain,    -   the human immunoglobulin K-polyadenylation (“poly A”) signal        sequence, and    -   the unique restriction sites AscI and FseI at the 5′- and        3′-end, respectively.

The plasmid map of the mAb CCR5 κ-light chain expression vector 4900 isshown in FIG. 1. The amino acid sequence of the mature (without signalsequence) mAb CCR5 κ-light chain is shown in SEQ ID NO:63.

b) Vector 4991

Vector 4991 is an expression plasmid for transient expression of a mAbCCR5 γ1-heavy chain (genomically organized expression cassette;exon-intron organization) in HEK293 cells.

Beside the mAb CCR5 γ1-heavy chain expression cassette this vectorcontains:

-   -   a hygromycin resistance gene as a selectable marker,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the mAb CCR5 γ1-heavy chain is composed of thefollowing elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   a synthetic 5′-untranslated region,    -   a murine immunoglobulin heavy chain signal sequence including a        signal sequence intron (signal sequence 1, intron, signal        sequence 2 [L1-intron-L2]),    -   the murine anti-CCR5 antibody mature variable heavy chain        encoding segment arranged with a unique BsmI restriction site at        the 5′-end (L2 signal sequence) and a splice donor site and a        unique NotI restriction site at the 3′-end,    -   a human/mouse heavy chain hybrid intron 2 including the mouse        heavy chain enhancer element (part JH₃, JH₄) (Neuberger, M. S.,        EMBO J. 2 (1983) 1373-1378),    -   the genomic human γ1-heavy gene constant domains,    -   the human γ1-immunoglobulin polyadenylation (“poly A”) signal        sequence, and    -   the unique restriction sites AscI and SgrAI at the 5′- and        3′-end, respectively.

The plasmid map of the mAb CCR5 γ1-heavy chain expression vector 4901 isshown in FIG. 2. The amino acid sequence of the mature (without signalsequence) mAb CCR5 γ1-heavy chain is shown in SEQ ID NO:64.

c) Vector 4995

Vector 4995 is an expression plasmid for transient expression of achimeric peptide-anti-CCR5 antibody γ1-heavy chain conjugate(genomically organized expression cassette; exon-intron organization) inHEK293 cells.

The vector 4995 is derived from plasmid 4991 in that way that the mAbCCR5 γ1-heavy chain is joint at the C-terminus with a nucleic acidencoding the antifusogenic peptide T-2635 (SEQ ID NO:33) and thepeptidic linker sequence [GQ₄]₃GNN (SEQ ID NO:40).

Beside the chimeric peptide anti-CCR5 antibody γ1-heavy chain conjugateexpression cassette this vector contains:

-   -   a hygromycin resistance gene as a selectable marker,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a beta(β)-lactamase gene which confers ampicillin resistance        in E. coli.

The transcription unit of the chimeric peptide-anti-CCR5 antibodyγ1-heavy chain conjugate is composed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   a synthetic 5′-untranslated region,    -   a murine immunoglobulin heavy chain signal sequence including a        signal sequence intron (signal sequence 1, intron, signal        sequence 2 [L1-intron-L2]),    -   the murine anti-CCR5 antibody mature variable heavy chain        encoding segment arranged with a unique BsmI restriction site at        the 5′-end (L2 signal sequence) and a splice donor site and a        unique NotI restriction site at the 3′-end,

a human/mouse heavy chain hybrid intron 2 including the mouse heavychain enhancer element (part JH₃, JH₄) (Neuberger, M. S., EMBO J. 2(1983) 1373-1378),

-   -   the genomic human γ1-heavy gene constant domains,    -   the antifusogenic peptide T-2635,    -   the peptidic linker sequence [GQ₄]₃GNN,    -   the human γ1-immunoglobulin polyadenylation (“poly A”) signal        sequence, and    -   the unique restriction sites AscI and SgrAI at the 5′- and        3′-end, respectively.

The plasmid map of the mAb CCR5 γ1-heavy chain conjugate expressionvector 4995 is shown in FIG. 3. The amino acid sequence of the mature(without signal sequence) conjugate heavy chain is shown in SEQ IDNO:65.

Example 2 Making of the Final Expression Plasmids

The fusion genes (heavy and/or light chain antibody fusion genes)comprising a mAb CCR5 gene segment, an optional linker gene segment andan antifusogenic peptide gene segment have been assembled with knownrecombinant methods and techniques by connection of the accordingnucleic acid segments. The nucleic acid sequences encoding the peptidiclinkers and antifusogenic polypeptides were each synthesized by chemicalsynthesis and then ligated into an E. coli plasmid for amplification.The subcloned nucleic acid sequences were verified by DNA sequencing.

Example 3 Transient Expression of Immunoglobulins and ImmunoglobulinVariants in HEK293 EBNA Cells

Recombinant anti-CCR5 antibodies and anti-CCR5 antibody-variants weregenerated by transient transfection of adherent growing HEK293-EBNAcells (human embryonic kidney cell line 293 expressingEpstein-Barr-Virus nuclear antigen; American type culture collectiondeposit number ATCC # CRL-10852) cultivated in DMEM (Dulbecco's modifiedEagle's medium, Gibco) supplemented with 10% ultra-low IgG FCS (fetalcalf serum, Gibco), 2 mM Glutamine (Gibco), 1% volume by volume (v/v)nonessential amino acids (Gibco) and 250 μg/ml G418 (Roche MolecularBiochemicals). For transfection FuGENE™ 6 Transfection Reagent (RocheMolecular Biochemicals) was used in a ratio of reagent (μl) to DNA (μg)ranging from 3:1 to 6:1. Light and heavy chains including antifusogenicpeptide-anti-CCR5 antibody conjugate light and heavy chains wereexpressed from two different plasmids using a molar ratio of light chainto heavy chain encoding plasmid ranging from 1:2 to 2:1, respectively.Antifusogenic peptide-anti-CCR5 antibody conjugates containing cellculture supernatants were harvested at day 4 to 11 after transfection.General information regarding the recombinant expression of humanimmunoglobulins in e.g. HEK293 cells is given in P. Meissner et al.,Biotechnol. Bioeng. (2001) 75:197-203.

Example 4 Expression Analysis using SDS PAGE, Western Blotting Transferand Detection with Immunoglobulin Specific Antibody Conjugates

The expressed and secreted antifusogenic peptide-anti-CCR5 antibodyconjugates were processed by sodium dodecyl sulfate (SDS) polyacrylamidegel electrophoresis (SDS-PAGE), and the separated anti-CCR5-antibody andantifusogenic peptide-anti-CCR5-antibody-conjugate chains weretransferred to a membrane from the gel and subsequently detected by animmunological method.

SDS-PAGE

LDS sample buffer, fourfold concentrate (4×): 4 g glycerol, 0.682 gTRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS (lithium dodecylsulfate), 0.006 g EDTA (ethylene diamin tetra acid), 0.75 ml of a 1% byweight (w/w) solution of Serva Blue G250 in water, 0.75 ml of a 1% byweight (w/w) solution of phenol red, add water to make a total volume of10 ml.

The culture broth containing the secreted antifusogenicpeptide-anti-CCR5 antibody conjugate was centrifuged to remove cells andcell debris. An aliquot of the clarified supernatant was admixed with ¼volumes (v/v) of 4xLDS sample buffer and 1/10 volume (v/v) of 0.5 M1,4-dithiotreitol (DTT). Then the samples were incubated for 10 min. at70° C. and protein separated by SDS-PAGE. The NuPAGE® Pre-Cast gelsystem (Invitrogen) was used according to the manufacturer'sinstruction. In particular, 10% NuPAGE® Novex® Bis-TRIS Pre-Cast gels(pH 6.4) and a NuPAGE® MOPS running buffer was used.

Western Blot

Transfer buffer: 39 mM glycine, 48 mM TRIS-hydrochloride, 0.04% byweight (w/w) SDS, and 20% by volume methanol (v/v).

After SDS-PAGE the separated antifusogenic peptide-anti-CCR5 antibodyconjugate chains were transferred electrophoretically to anitrocellulose filter membrane (pore size: 0.45 μm) according to the“Semidry-Blotting-Method” of Burnette (W. N. Burnette, Anal. Biochem.(1981) 112:195-203).

Immunological Detection

TBS-buffer: 50 mM TRIS-hydrochloride, 150 mM NaCl, adjusted to pH 7.5Blocking solution: 1% (w/v) Western Blocking Reagent (Roche MolecularBiochemicals) in TBS-buffer

TBST-Buffer: 1×TBS-buffer with 0.05% by volume (v/v) Tween-20 Forimmunological detection the western blotting membranes were incubatedwith shaking at room temperature two times for 5 minutes in TBS-bufferand once for 90 minutes in blocking solution.

Detection of the Peptide Immunoglobulin Conjugate Chains

Heavy chain: For detection of the heavy chain of the antifusogenicpeptide-anti-CCR5 antibody conjugate a purified rabbit anti-human IgGantibody conjugated to a peroxidase was used (DAKO, Code No. P 0214).

Light chain: The light chain of the antifusogenic peptide-anti-CCR5antibody conjugate was detected with a purified peroxidase conjugatedrabbit anti-human kappa light chain antibody (DAKO, Code No. P 0129).

For visualization of the antibody light and heavy chains washed andblocked Western blot membranes were first incubated in case of a heavychain with a purified rabbit anti-human IgG antibody conjugated to aperoxidase or in case of a light chain with a purified peroxidaseconjugated rabbit anti-human kappa light chain antibody in a 1:10,000dilution in 10 ml blocking solution at 4° C. with shaking over night.After washing the membranes three times with TBTS-buffer and once withTBS buffer for 10 min. at room temperature, the Western-blot membraneswere developed with a Luminol/peroxid-solution generatingchemiluminescence (Lumi-Light^(PLUS) Western Blotting Substrate, RocheMolecular Biochemicals). Therefore the membranes were incubated in 10 mlLuminol/peroxid-solution for 10 seconds to 5 minutes and the emittedlight was detected afterwards with a LUMI-Imager F1 Analysator (RocheMolecular Biochemicals) and/or was recorded with an x-ray-film. Theintensity of the spots was quantified with the LumiAnalyst Software(Version 3.1).

Multiple-Staining of Immunoblots

The secondary peroxidase-labeled antibody conjugate used for thedetection can be removed from the stained blot by incubating themembrane for one hour at 70° C. in 1 M TRIS-hydrochloride-buffer (pH6.7) containing 100 mM beta-mercaptoethanol and 20% (w/v) SDS. Afterthis treatment the blot can be stained with a different secondaryantibody a second time. Prior to the second detection the blot is washedthree times at room temperature with shaking in TBS-buffer for 10minutes each.

Example 5 Affinity Purification, Dialysis and Concentration of PeptideImmunoglobulin Conjugates

The expressed and secreted antifusogenic peptide-anti-CCR5 antibodyconjugates were purified by affinity chromatography using ProteinA-Sepharose™ CL-4B (GE Healthcare former Amersham Bioscience, Sweden)according to known methods. Briefly, after centrifugation (10,000 g for10 minutes) and filtration through a 0.45 μm filter the peptideimmunoglobulin conjugate containing clarified culture supernatants wereapplied on a Protein A-Sepharose™ CL-4B column equilibrated with PBSbuffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4).Unbound proteins were washed out with PBS equilibration buffer and 0.1 Mcitrate buffer, pH 5.5. The antifusogenic peptide-anti-CCR5 antibodyconjugates were eluted with 0.1 M citrate buffer, pH 3.0, and theconjugate containing fractions were neutralized with 1 M TRIS-Base.Then, the antifusogenic peptide-anti-CCR5 antibody conjugates wereextensively dialyzed against PBS buffer at 4° C., concentrated with aUltrafree®-CL Centrifugal Filter Unit equipped with a Biomax-SK membrane(Millipore Corp., USA) and stored in an ice-water bath at 0° C. Theintegrity of the conjugates was analyzed by SDS-PAGE in the presence andabsence of a reducing agent and staining with Coomassie brilliant blueas described in example 4. Aggregation of antifusogenicpeptide-anti-CCR5 antibody conjugates was analyzed by analytical sizeexclusion chromatography.

Example 6 Deglycosylation of Peptide Immunoglobulin Conjugates

N-linked carbohydrates of anti-CCR5 antibodies and antifusogenicpeptide-anti-CCR5 antibody conjugates were cleaved off by enzymatictreatment with Peptide-N-Glycosidase F (PNGaseF, Roche MolecularBiochemicals, Mannheim, Germany or Prozyme, San Leandro, Calif.).Therefore, the anti-CCR5 antibodies and antifusogenic peptide-anti-CCR5antibody conjugates were incubated at 37° C. for 12-24 h using 50 mUPNGaseF per mg N-glycosylated protein in PBS buffer at a proteinconcentration of about 2 mg/ml. Thereafter the Peptide-N-Glycosidase Fwas separated by preparative gel filtration according to known methods.Briefly, PNGaseF treated anti-CCR5-antibodies and antifusogenicpeptide-anti-CCR5 antibody conjugates were applied on a Superose™ 1210/300 GL column (GE Healthcare former Amersham Bioscience, Sweden)equilibrated with PBS buffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCland 2.7 mM KCl, pH 7.4) and then eluted with equilibration buffer at aflow rate of 0.5-1.0 ml/min using the Äkta explorer chromatographysystem from Amersham Bioscience (GE Healthcare former AmershamBioscience, Sweden).

Example 7 Single-Cycle Antiviral Activity Assay

For the production of pseudotyped NL-Bal viruses, plasmid pNL4-3Δenv(HIV pNL4-3 genomic construct with a deletion within the env gene) andpcDNA3.1/NL-BAL env [pcDNA3.1 plasmid containing NL-Bal env gene(obtained from NIBSC Centralized Facility for AIDS Reagents)] wereco-transfected into the HEK 293FT cell line (Invitrogen), cultured inDulbecco's' modified minimum medium (DMEM) containing 10% fetal calfserum (FCS), 100 U/mL Penicillin, 100 μg/mL Streptomycin, 2 mML-glutamine and 0.5 mg/mL geniticin (all media from Invitrogen/Gibco).The supernatants containing pseudotyped viruses were harvested two daysfollowing transfection, and cellular debris was removed by filtrationthrough a 0.45 μm pore size PES (polyethersulfone) filter (Nalgene) andstored at −80° C. in aliquots. For normalization in assay performance,virus stock aliquots were used to infect JC53-BL (US NIH Aids ReagentProgram) cells yielding approximately 1.5×10⁵ RLU (relative light units)per well. Test antifusogenic peptide-anti-CCR5 antibody conjugates,reference antibodies and reference antifusogenic peptides (T-20, T-1249,T-651 and T-2635) were serially diluted in 96-well plates. The assay wascarried out in quadruplicates. Each plate contained cell control andvirus control wells. The equivalent of 1.5×10⁵ RLU of virus stocks wereadded to each well, then 2.5×10⁴ JC53-BL cells were added to each well,with a final assay volume of 200 μl per well. After 3 day incubation at37° C., 90% Relative Humidity, and 5% CO₂, media were aspirated and 50μl of Steady-Glo® Luciferase Assay System (Promega) was added to eachwell. The assay plates were read on a Luminometer (Luminoskan, ThermoElectron Corporation) after 10 minutes of incubation at roomtemperature. Percent inhibition of luciferase activity was calculatedfor each dose point after subtracting the background, and IC₅₀ andIC₉₀-values were determined by using XLfit curve fitting software forExcel (version 3.0.5 Build12; Microsoft). Results are shown in Table 3.

TABLE 3 Antiviral activity of antifusogenic polypeptides, antibodies andantifusogenic peptide-anti-CCR5 antibody conjugates Antiviral activityCompound IC₅₀ (ng/mL) IC₉₀ (ng/mL) Reference antibody 1 (inert) inactiveinactive Reference antibody 2 (inert) inactive inactive T-20 206 3955T-1249 11 90 T-651 11 139 T-2635 14 161 mAb CCR5 (4901/4900) 114 2387Chimeric peptide mAb CCR5 7 45 conjugate (4995/4900)

Example 8 Cell-Cell Fusion Assay

At day 1, gp160-expressing HeLa cells (2×10⁴ cells/50 μl/well) areseeded in a white 96 microtiter plate in DMEM medium supplemented with10% FCS and 2 μg/ml doxycycline. At day 2, 100 μl of supernatant sampleor antibody control per well is added in a clear 96 microtiter plate.Then 100 μl containing 8×10⁴ CEM-NKr-Luc suspension cells in medium areadded and incubated 30 min. at 37° C. The HeLa cell culture medium isaspirated from the 96 well plate, 100 μl from the 200 μlantibody/CEM-NKr-Luc mixture is added and incubated overnight at 37° C.At day 3, 100 μl/well Bright-Glo™ Luciferase assay substrate(1,4-dithiothreitol and sodium dithionite; Promega Corp., USA) is addedand luminescence is measured after a minimum of 15 min. incubation atRT.

Materials:

HeLa-R5-16 cells (cell line to express HIV gp160 upon doxycyclineinduction) are cultured in DMEM medium containing nutrients and 10% FCSwith 400 μg/ml G418 and 200 μg/ml Hygromycin B. CEM.NKR-CCR5-Luc(Catalog Number: 5198, a T-cell line available from NIH AIDS Research &Reference Reagent Program McKesson BioServices Corporation Germantown,Md. 20874, USA). Cell Type: CEM.NKR-CCR5 (Cat. #4376) is transfected(electroporation) to express the luciferase gene under thetranscriptional control of the HIV-2 LTR and propagated in RPMI 1640containing 10% fetal bovine serum, 4 mM glutamine,penicillin/streptomycin (100 U/mL Penicillin, 100 μg/mL Streptomycin),and 0.8 mg/ml geniticin sulfate (G418). Growth Characteristics: Roundlymphoid cells, morphology not very variable. Cells grow in suspensionas single cells, which can form small clumps. Split 1:10 twice weekly.Special Characteristics: Express luciferase activity aftertransactivation of the HIV-2 LTR. Suitable for infection with primaryHIV isolates, for neutralization and drug-sensitivity assays (C.Spenlehauer et al., Virology (2001) 280:292-300; A. Trkola et al., J.Virol. (1999) 73:8966-74). The cell line was obtained through the NIHAIDS Research and Reference Reagent Program, NIAID, NIH from Drs. JohnMoore and Catherine Spenlehauer. Bright-Glo™ Luciferase assay buffer(Promega Corp. USA, Part No E2264B), Bright-Glo™, Luciferase assaysubstrate (Promega Corp. USA, part No EE26B).

Example 9 Antiviral Activity Assay in Peripheral Blood Mononuclear Cells(PBMC)

Human PBMC are isolated from buffy-coats (obtained from the StanfordBlood Center) by a Ficoll-Paque (Amersham, Piscataway, N.J., USA)density gradient centrifugation according to manufacturer's protocol.Briefly, blood is transferred from the buffy coats in 50 ml conicaltubes and diluted with sterile Dulbecco's phosphate buffered saline(Invitrogen/Gibco) to a final volume of 50 ml. Twenty-five ml of thediluted blood are transferred to two 50 ml conical tubes, carefullyunderlayered with 12.5 ml of Ficoll-Paque Plus (Amersham Biosciences)and centrifuged at room temperature for 20 min. at 450×g withoutbraking. The white cell layer is carefully transferred to a new 50 mlconical tube and washed twice with PBS. To remove remaining red bloodcells, cells are incubated for 5 min. at room temperature with ACK lysisbuffer (Biosource) and washed one more time with PBS. PBMC are countedand incubated at a concentration of 2-4×10⁶ cells/ml in RPMI1640containing 10% FCS (Invitrogen/Gibco), 1% penicillin/streptomycin, 2 mML-glutamine, 1 mM sodium-pyruvate, and 2 μg/ml Phytohemagglutinin(Invitrogen) for 24 h at 37° C. Cells are incubated with 5 Units/mlhuman IL-2 (Roche Molecular Biochemicals) for a minimum of 48 h prior tothe assay. In a 96 well round bottom plate, 1×10⁵ PBMC are infected withthe HIV-1 JR-CSF virus (Y. Koyanagi et al., Science (1987) 236:819-22)in the presence of serially diluted testpeptide-immunoglobulin-conjugates, reference immunoglobulins andreference peptides (T-20, T-1249, T-651 and T-2635). The amount of virusused is equivalent to 1.2 ng HIV-1 p24 antigen/well. Infections are setup in quadruplicates. Plates are incubated for 6 days at 37° C. Virusproduction is measured at the end of infection by using p24 ELISA (HIV-1p24 ELISA #NEK050B, Perkin Elmer/NEN) using the sigmoid dose-responsemodel with one binding site in Microsoft Excel Fit (version 3.0.5 Build12; equation 205; Microsoft).

1. A conjugate comprising an anti-CCR5 antibody (mAb CCR5) havingtermini comprising an N-terminus and a C-terminus; and an antifusogenicpeptide, conjugated to said N-terminus or said C-terminus.
 2. Theconjugate of claim 1, comprising two antifusogenic peptides, conjugatedto said anti-CCR5 antibody termini.
 3. The conjugate according to claim1, wherein said antifusogenic peptides are linear peptides that comprisean amino acid sequence of from 5 to 100 amino acids.
 4. The conjugate ofclaim 1, wherein said antifusogenic peptide is conjugated to saidN-terminus or C-terminus through a peptide linker.
 5. The conjugate ofclaim 1, wherein said mAb CCR5 comprises two heavy chains and two lightchains.
 6. The conjugate of claim 5, characterized by the generalformula mAb CCR5-[linker]_(m)-[antifusogenic peptide]_(n), wherein m isindependently for each antifusogenic peptide either 0 or 1, and n is aninteger of from 1 to
 8. 7. The conjugate of claim 6, comprising aconjugate of heavy and/or light chain of mAb CCR5 and an antifusogenicpeptide(s) (“chain conjugate”), selected from the group consisting of:(1) [antifusogenic peptide]-[linker]_(m)-[heavy chain]; (2) [heavychain]-[linker]_(m)-[antifusogenic peptide]; (3) [antifusogenicpeptide]-[linker]_(m)-[heavy chain]-[antifusogenic peptide]; (4)[antifusogenic peptide]-[linker]_(m)-[light chain]; (5) [lightchain]-[linker]_(m)-[antifusogenic peptide]; (6) [antifusogenicpeptide]-[linker]_(m)-[light chain]-[antifusogenic peptide]; (7)[antifusogenic peptide]-[linker]_(m)-[heavychain]-[linker]_(m)-[antifusogenic peptide]; and (8) [antifusogenicpeptide]-[linker]_(m)-[light chain]-[linker]_(m)-[antifusogenicpeptide], wherein each linker can be the same or different, wherein eachm is independently an integer of 1 or
 0. 8. The conjugate of claim 7,which comprises a chain conjugate (2), (3), (4), or (7).
 9. Theconjugate of claim 8, which comprises: 2×[mAb CCR5 light chain] and2×(2), 2×[mAb CCR5 light chain] and 2×(3), 2×[mAb CCR5 heavy chain] and2×(4), or 2×[mAb CCR5 light chain] and 2×(7).
 10. The conjugate of claim1, wherein said antifusogenic peptide has a sequence selected from thegroup SEQ ID NO:29 to
 35. 11. The conjugate according of claim 1,wherein said anti-CCR5 antibody comprises a variable heavy chain domainconsisting of an immunoglobulin framework and a CDR3 region selectedfrom the group consisting of the heavy chain CDR3 sequences SEQ ID NO:16and SEQ ID NO:17.
 12. The conjugate of claim 1, wherein said anti-CCR5antibody comprises a variable heavy chain domain consisting of animmunoglobulin framework and a CDR3 region selected from the groupconsisting of heavy chain CDR3 sequences SEQ ID NOS:16 and 17; a CDR2region selected from the group consisting of heavy chain CDR2 sequencesSEQ ID NOS: 13, 14, and 15; and a CDR1 region selected from the groupconsisting of heavy chain CDR1 sequences SEQ ID NOS:9, 10, 11, and 12.13. The conjugate of claim 1, wherein said anti-CCR5 antibody comprisesa heavy chain variable domain selected from the group consisting of SEQID NOS:1, 3, 5, and
 7. 14. The conjugate of claim 1, wherein saidanti-CCR5 antibody comprises a variable light chain domain consisting ofan immunoglobulin framework and a CDR1 region selected from SEQ IDNOS:18, 19, and 20; a CDR2 region selected from SEQ ID NOS:21, 22, and23; and a CDR3 region selected from SEQ ID NOS:24 and
 25. 15. Theconjugate of claim 12, wherein said anti-CCR5 antibody comprises asheavy chain CDRs the CDRs of SEQ ID NO: 1 and as light chain CDRs theCDRs of SEQ ID NO: 2, as heavy chain CDRs the CDRs of SEQ ID NO: 3 andas light chain CDRs the CDRs of SEQ ID NO: 4, as heavy chain CDRs theCDRs of SEQ ID NO: 5 and as light chain CDRs the CDRs of SEQ ID NO: 6,or as heavy chain CDRs the CDRs of SEQ ID NO: 7 and as light chain CDRsthe CDRs of SEQ ID NO:
 8. 16. The conjugate of claim 15, wherein saidanti-CCR5 antibody comprises a variable heavy chain domain and avariable light chain domain independently selected from the groupconsisting of a) the heavy chain (V_(H)) variable domain defined byamino acid sequence SEQ ID NO:1 and the light chain (V_(L)) variabledomain defined by SEQ ID NO:2; b) the heavy chain variable domaindefined by amino acid sequence SEQ ID NO:3 and the light chain variabledomain defined by SEQ ID NO:4; c) the heavy chain variable domaindefined by amino acid sequence SEQ ID NO:5 and the light chain variabledomain defined by SEQ ID NO:6; d) the heavy chain variable domaindefined by amino acid sequence SEQ ID NO:7 and the light chain variabledomain defined by SEQ ID NO:8.
 17. The conjugate of claim 1, whereinsaid conjugate comprises: said anti-CCR5 antibody, selected from thegroup consisting of the heavy chain (V_(H)) variable domain defined byamino acid sequence SEQ ID NO: 1 and the light chain (V_(L)) variabledomain defined by SEQ ID NO:2; the heavy chain variable domain definedby amino acid sequence SEQ ID NO:3 and the light chain variable domaindefined by amino acid SEQ ID NO:4; the heavy chain variable domaindefined by amino acid sequence SEQ ID NO:5 and the light chain variabledomain defined by amino acid SEQ ID NO:6; and the heavy chain variabledomain defined by amino acid sequence SEQ ID NO:7 and the light chainvariable domain defined by amino acid SEQ ID NO:8; a linker selectedfrom the group consisting of amino acids glycine (G) and asparagine (N),the tripeptide GST, and SEQ ID NO: 36-62; and an antifusogenic peptidehaving a sequence selected from the group consisting of SEQ ID NO:29 to35.
 18. The conjugate of claim 1, wherein said antifusogenic peptide hasa sequence selected from the group consisting of SEQ ID NOS:29 to 35.19. The conjugate of claim 17, wherein said conjugate comprises twolight chain variable domains of SEQ ID NO:2, two conjugates of type (2)each comprising a heavy chain variable domain of SEQ ID NO:1, a linkerof SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, incomprising two light chain variable domains of SEQ ID NO:4, twoconjugates of type (2) each comprising a heavy chain variable domain ofSEQ ID NO:3, a linker of SEQ ID NO:40 and an antifusogenic peptide ofSEQ ID NO:33, in comprising two light chain variable domains of SEQ IDNO:6, two conjugates of type (2) each comprising a heavy chain variabledomain of SEQ ID NO:5, a linker of SEQ ID NO:40 and an antifusogenicpeptide of SEQ ID NO:33, or in comprising two light chain variabledomains of SEQ ID NO:8, two conjugates of type (2) each comprising aheavy chain variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 andan antifusogenic peptide of SEQ ID NO:33.
 20. The conjugate of claim 1,wherein said anti-CCR5 antibody is of IgG4 subclass, or is of IgG1 orIgG2 subclass with a mutation in amino acid S228, L234, L235, and/orD265, and/or contains the PVA236 mutation
 21. A conjugate according toclaim 20, wherein said anti-CCR5 antibody of IgG4 subclass has themutation S228P and said anti-CCR5 antibody of IgG1 subclass has themutations L234A and L235A.
 22. A method producing a conjugate of claim1, wherein said method comprises: a) cultivating a cell containingnucleic acid molecules encoding a conjugate according to claim 1 underconditions suitable for the expression of the conjugate; and b)recovering the conjugate from the cell or the cell culture supernatant.23. A pharmaceutical composition, comprising an effective amount of aconjugate of claim 1, together with a pharmaceutically acceptableexcipient or carrier.
 24. A method for treating a viral infection,comprising: administering an effective amount of a conjugate of claim 1to a subject in need thereof.
 25. The method of claim 24, wherein theviral infection is HIV infection.